Fluorescence has long been recognized as an important tool for probing biological structure and function. Development of optically active structures that can enhance fluorescence intensity have gained much attention as a means for detecting fluorescent-tagged analytes at low concentrations for applications in DNA expression analysis and protein diagnostic assays. The majority of structures developed to date for this purpose utilize plasmonics of metals to increase the excitation of fluorophores through enhanced near fields, to increase the quantum yield by increasing the intrinsic radiative decay rate of the fluorophores, to increase the directional emission or to employ some combination of these processes. Background references include Y. J. Hung, I. I. Smolyaninov, C. C. Davis, and H. C. Wu, “Fluorescence enhancement by surface gratings,” Optics Express, vol. 14, pp. 10825-10830, 2006; T. Hayakawa, S. T. Selvan, and M. Nogami, “Field enhancement effect of small Ag particles on the fluorescence from Eu3+-doped SiO2 glass,” Applied Physics Letters, vol. 74, pp. 1513-1515, 1999; K. Aslan, S. N. Malyn, and C. D. Geddes, “Metal-enhanced fluorescence from gold surfaces: Angular dependent emission,” Journal of Fluorescence, vol. 17, pp. 7-13, 2007; K. Aslan, I. Gryczynski, J. Malicka, E. Matveeva, J. R. Lakowicz, and C. D. Geddes, “Metal-enhanced fluorescence: an emerging tool in biotechnology,” Current Opinion in Biotechnology, vol. 16, pp. 55-62, 2005. Another reference of interest is W. Budach, D. Neuschafer, C. Wanke, and S. D. Chibout, “Generation of transducers for fluorescence-based microarrays with enhanced sensitivity and their application for gene expression profiling,” Analytical Chemistry, vol. 75, pp. 2571-2577, 2003 which discloses dielectric gratings for detection of fluorescence-labeled samples.
Recently, photonic crystal (PC) sensors have also been used to enhance the emission intensity of fluorophores and quantum dots. Photonic crystals, also commonly referred to as photonic bandgap structures, are periodic dielectric structures exhibiting a spatially periodic variation in refractive index that forbids propagation of certain frequencies of incident electromagnetic radiation. The photonic band gap of a photonic crystal refers to the range of frequencies of electromagnetic radiation for which propagation through the structure is prevented. The photonic band gap phenomenon may be conceptualized as complete reflection of incident electromagnetic radiation having selected frequencies due to interaction with the periodic structural domains of a photonic crystal. The spatial arrangement and refractive indices of these structural domains generate photonic bands gaps that inhibit propagation of electromagnetic radiation centered about a particular frequency.
Photonic crystals provide an electromagnetic analog to electron-wave behavior observed in crystals wherein electron-wave concepts, such as dispersion relations, Bloch wave functions, van Hove singularities and tunneling, having electromagnetic counterparts in photonic crystals. In semiconductor crystals, for example, an electronic band gap of energy states for which electrons are forbidden results from a periodic atomic crystalline structure. By analogy, in a photonic crystal, a photonic band gap of forbidden energies (or wavelengths/frequencies) of electromagnetic radiation results from a periodic structure of a dielectric material, where the periodicity is of a distance suitable to interact with incident electromagnetic radiation.
Selection of the physical dimensions, refractive indices and spatial distribution of structural domains of a photonic crystal provides an effective means of designing a photonic crystal a photonic band gap with a selected frequency distribution. One-dimensional, two-dimensional and three-dimensional photonic crystals have been fabricated providing complete or at least partial photonic band having selected frequency distributions gaps in one or more directions and/or polarizations of light. Photonic crystals have also been fabricated having selected local disruptions (e.g., missing or differently-shaped portions of the structural domains of periodic array) in their periodic structure, thereby generating defect cavity modes with frequencies within a forbidden bandgap of the crystal. (See Cunningham, U.S. Pat. No. 6,990,259). Photonic crystals having specific defects are of particular interest because they provide optical properties useful for controlling and manipulating electromagnetic radiation, such as the ability to provide optical confinement and/or wave guiding with very little, or substantially no, radiative losses.
As diffraction and optical interference processes give rise to the photonic band gap phenomenon, the periodicity of photonic crystal structures is typically on the order of the wavelength of incident electromagnetic radiation. Accordingly, photonic crystals for controlling and manipulating visible and ultraviolet electromagnetic radiation typically comprise dielectric structures with periodic structural domains having submicron physical dimensions on the order of 100s of nanometers. A number of fabrication pathways for making periodic structures having these physical dimensions have been developed over the last decade, including micromachining and nanomachining techniques (e.g., lithographic patterning and dry/wet etching, electrochemical processing etc.), colloidal self assembly, replica molding, layer-by-layer assembly and interference lithography. Advances in these fabrication techniques have enabled fabrication of one-dimensional, two-dimensional and three-dimensional photonic crystals from a range of materials including dielectric crystals, polymers and colloidal materials.
Because PC sensors are comprised of dielectric materials, they will not quench fluorophores within <30 nm of their surface by resonant energy transfer, and they can exhibit high Q-factors due to their low absorption loss. Typically comprised of a one-dimensional (1D) or two-dimensional (2D) periodic surface structure formed from a low refractive index (RI) dielectric material that is overcoated with a high RI thin film, these devices can be fabricated upon plastic substrates inexpensively over large areas by a nanoreplica molding process and incorporated into the surface of glass slides, microfluidic channels, and microtiter plates. The device period, grating depth, film thicknesses, and RIs of the materials are chosen in such a way that the PCs can support guided-mode resonances at designated wavelengths, where the device reflects ˜100% of incident light at the resonant wavelengths with all other wavelengths being transmitted. Under resonant conditions, excited leaky modes are localized in space during their finite lifetimes, which enhances the near electric-field intensity of the PC structure and thus enhances the excitation of fluorophores near the PC surface.
A representative photonic crystal sensor having a one dimensional periodic surface grating structure and its associated detection instrument is shown in FIG. 1. The PC sensor 1 consists of a substrate polyester sheet 10, a periodic surface grating structure 12 formed on the substrate 10 in the form of alternating high and low regions, and a high index of refraction (TiO2) material 14 deposited on the grating structure 12. The device is interrogated with white light from a light source (not shown) that is coupled to an illuminating fiber 16 of a fiber optic probe 18. The illuminating light is passed through a collimating lens 20 and is incident on the PC sensor 1. A narrow band of reflected light is captured by a detecting fiber 26 of the probe 18. The reflected light is passed to a spectrometer 28. Binding of a sample in a sample medium (which may be air or water) causes a shift in the peak wavelength value of the reflected light, with the amount of the shift being a measure of the amount of binding to the surface of the sensor.
Further background information relating to photonic crystals sensors and their properties and methods of manufacture are disclosed in the following references, which are incorporated by reference herein: P. C. Mathias, N. Ganesh, L. L. Chan, and B. T. Cunningham, “Combined enhanced fluorescence and label-free biomolecular detection with a photonic crystal surface,” Applied Optics, vol. 46, pp. 2351-2360, 2007; N. Ganesh, W. Zhang, P. C. Mathias, E. Chow, J. A. N. T. Soares, V. Malyarchuk, A. D. Smith, and B. T. Cunningham, “Enhanced fluorescence emission from quantum dots on a photonic crystal surface,” Nature Nanotechnology, vol. 2, pp. 515-520, 2007; N. Ganesh and B. T. Cunningham, “Photonic-crystal near-ultraviolet reflectance filters fabricated by nanoreplica molding,” Applied Physics Letters, vol. 88, 2006; C. J. Choi and B. T. Cunningham, “Single-step fabrication and characterization of photonic crystal biosensors with polymer microfluidic channels,” Lab on a Chip, vol. 6, pp. 1373-1380, 2006; B. T. Cunningham, P. Li, S. Schulz, B. Lin, C. Baird, J. Gerstenmaier, C. Genick, F. Wang, E. Fine, and L. Laing, “Label-free assays on the BIND system,” Journal of Biomolecular Screening, vol. 9, pp. 481-490, 2004; S. S. Wang, R. Magnusson, J. S. Bagby, and M. G. Moharam, “Guided-Mode Resonances in Planar Dielectric-Layer Diffraction Gratings,” Journal of the Optical Society of America a-Optics Image Science and Vision, vol. 7, pp. 1470-1474, 1990; R. Magnusson and S. S. Wang, “New Principle for Optical Filters,” Applied Physics Letters, vol. 61, pp. 1022-1024, 1992; S. S. Wang and R. Magnusson, “Theory and Applications of Guided-Mode Resonance Filters,” Applied Optics, vol. 32, pp. 2606-2613, 199; C. Y. Wei, S. J. Liu, D. G. Deng, J. Shen, J. D. Shao, and Z. X. Fan, “Electric field enhancement in guided-mode resonance filters,” Optics Letters, vol. 31, pp. 1223-1225, 2006.
Given substantial advances in their fabrication and their unique optical properties, photonic crystal-based sensors have been recently developed for a range of biosensing applications. To operate as a biosensor, a photonic crystal is provided in a configuration such that its active area is exposed to a fluid containing analytes for detection. The presence of analyte proximate to the photonic crystal sensor modulates the resonant coupling of light into the crystal, thereby resulting in a measurable change in the wavelength distribution of electromagnetic radiation transmitted, scattered or reflected by the crystal resulting from changes in the photonic band gap of the crystal. The highly localized nature of the confined electromagnetic field generated by the crystal ensures that that detection via photonic crystal based sensors is restricted to a probe region proximate to the active area of the sensor (that is, generally less than 400 nm from the surface). In typical sensing applications, a read out system is used wherein polarized electromagnetic radiation having a selected wavelength distribution is provided to the photonic crystal and subsequently reflected or transmitted electromagnetic radiation is frequency analyzed by an appropriate photodetector, such as a spectrometer in combination with an appropriate detector. By observing and/or quantifying the change in wavelength distribution resulting from interaction of the fluid and the photonic crystal, analytes in the probe region are detected and/or analyzed. Biosensors incorporating photonic crystal structures are described in the following references which are hereby incorporated by reference in their entireties: U.S. Pat. Nos. 7,118,710, 7,094,595, and 6,990,259; U.S. Published applications 2007/0009968; 2002/0127565; 2003/0059855; 2007/0009380; 2003/0027327; and Cunningham, B. T., P. Li, B. Lin and J. Pepper, Colorimetric Resonant Reflection as a Direct Biochemical Assay Technique, Sensor and Actuators B, 2002, 81, pgs 316-328; and Cunningham, B. T. J. Qiu, P. Li, J. Pepper and B. Hugh, A Plastic Calorimetric Resonant Optical Biosensor for Multiparallel Detection of Label Free Biochemical Interactions, Sensors and Actuators B, 2002, 85, pgs 219-226.
Advantages provided by photonic crystals for biosensing include the ability to detect and characterize a wide range of materials, including peptides, proteins, oligonucleotides, cells, bacteria and virus particles, without the use of labels, such as fluorescent labels and radioligands, or secondary reporter systems. Direct detection provided by photonic crystal sensing enhances ease of implementation of these techniques by eliminating labor intensive processing required to synthetically link and/or read out a label or reporter system. This beneficial aspect of photonic crystal-based sensing also eliminates a significant source of experimental uncertainty arising from the influence of a label or reporter system on molecular conformation, reactivity, bioactivity and/or kinetics; and eliminates problems arising from liquid phase fluorescence quenching processes. Photonic crystal based sensors are also compatible with functionalization, for example by incorporation of biomolecules and/or candidate therapeutic molecules bound to the surface of the active area of the photonic crystal structure; a capability which is particularly attractive for selectively detecting specific target molecules for screening and biosensing applications. Other benefits provided by photonic crystal approaches to biosensing include: (i) good sensitivity and image resolution; (ii) compatibility with relatively straightforward optical readout systems, (iii) and the ability to provide highly localized detection useful for multichannel systems having a high area density of independent sensor channels. As a result of these attributes, photonic crystal based sensors are emerging as a major tool for selective biochemical detection and analysis in diverse fields including genomics, proteomics, pharmaceutical screening and biomedical diagnostics.
Even with these advances, the sensitivity of the sensors limits their applications. Development of sensor designs that enhance sensitivity is especially important because it allows detection of lower concentration of analytes and detection of small molecules with higher signal-to-noise ratio.
Previously, enhancement of optical biosensor sensitivity has been achieved through the use of polymer hydrogels such as dextran to extend the surface area for ligand attachment into a 3-dimensional volume within the evanescent field region of surface plasma resonance (SPR) sensors. Although hydrogel films offer a high surface area for covalent attachment of biomolecules, disadvantages of this method include (1) the hydrogel film is not an integral part of the device, (2) it involves a complex procedure for deposition and functionalization using liquid-based processes, and (3) as a polymer based on a sugar monomer, the dextran layer is subject to swelling and/or dissociation by extremes in pH. Therefore, enhancement of biosensor surface area using a more chemically and mechanically robust system is needed.
Glancing angle deposition (GLAD) is a physical vapor deposition technique in which the angle between the incoming flux and the surface of the substrate is set to be typically less than 15°. The technique is described in the following references, incorporated by reference herein: J. G. W. v. d. Waterbeemd and G. W. v. Oosterhout, “Effect of the Mobility of Metal Atoms on the Structure of Thin Films Deposited at Oblique Incidence,” Philips Res. Rep., vol. 22, pp. 375-387, 1967; K. Robbie, L. J. Friedrich, S. K. Dew, T. Smy, and M. J. Brett, “Fabrication of Thin-Films with Highly Porous Microstructures,” Journal of Vacuum Science & Technology a-Vacuum Surfaces and Films, vol. 13, pp. 1032-1035, 1995. L. Abelmann and C. Lodder, “Oblique evaporation and surface diffusion,” Thin Solid Films, vol. 305, pp. 1-21, 1997.
Embodiments described below include biosensors with nanorod structures to enhance the surface area of the PC sensor, with the nanorod structures created using the GLAD technique.